The dopamine transporter (DAT) plays a crucial role in the regulation of brain dopamine (DA) homeostasis. Through re-uptake of DA, DAT serves two important functions: the termination of synaptic transmission at dopaminergic terminals, and the replenishment of vesicular DA pools. In addition to uptake or direct transport, DAT can also function to release DA. This process, which is referred to as reverse transport or efflux, is the mechanism used by potent and highly addictive psychostimulants, such as amphetamine and its analogues, to increase extracellular DA levels in motivational and reward areas of the brain. It has long being recognized that DA neurons release DA through exocytotic and non-exocytotic processes. However, the exact mechanism by which physiological signals or psychostimulants, such as amphetamine, induce DA release through DAT still remains a complex and not completely understood area of research. Thus, examining the basic mechanism(s) that affect reverse transport through DAT is critical for both understanding fundamental aspects of DA regulation and clinical intervention in DA-related brain disorders associated with the therapeutic use and abuse of psychostimulants. The long-term goal of our research program is to identify and characterize signaling mechanisms that control DA release through DAT, and elucidate the molecular actions of psychostimulants. This application is based on our recent discovery that the beta upsilon subunit of G proteins (Gbetagamma) binds DAT and regulates transporter activity. This effect was demonstrated in cultured cells, brain synaptosomes, and in vivo. More importantly, activation of Gbetagamma promotes DAT-mediated DA efflux, whereas inhibition of Gbetagamma attenuates amphetamine-elicited DA efflux in cultured cells. Finally, activation of Gbetagamma enhances whereas inhibition of Gbetagamma reduces amphetamine-evoked locomotor activity in vivo. Based on these preliminary data, the central hypothesis of this proposal is that the interaction between DAT and beta upsilon subunits promotes DA release through DAT and is involved in the actions of amphetamine. In this proposal we will i) identify the Gbetagamma interaction site(s) in DAT and their role in transporter regulation, ii) test the hypothesis that Gbetagamma is involved in DAT-mediated DA efflux, and iii) test the hypothesis that Gbetagamma is involved in amphetamine's actions in vivo. The successful completion of the studies proposed here will provide a detailed characterization of the DAT-Gbetagamma interaction and a clear understanding of its contribution to DAT reverse transport. The fact that amphetamine induces DAT reversal suggests that DA can also be released through DAT under physiological conditions. Therefore, our proposed studies will define not only the role that Gbetagamma subunits play in the addictive properties of amphetamine, but also the contribution of Gbetagamma subunits to DA homeostasis as we grow our current understanding of the molecular details underlying physiological DAT reverse transport. The long-term goal of our research program is to identify novel therapeutic targets that can be used in the treatment of neuropsychiatric disorders, including drug addiction.